Abstract

EBV-encoded latent membrane protein 1 (LMP1) has oncogenic potential and is expressed in many EBV-associated malignancies. Although LMP1 is regarded as a potential tumor-associated antigen for immunotherapy and several LMP1-specific MHC class I–restricted CTL epitopes have been reported, little is known regarding MHC class II–restricted CD4 helper T-lymphocyte (HTL) epitopes for LMP1. The goal of the present studies was to determine whether MHC class II–restricted CD4 T-cell responses could be induced against the LMP1 antigen and to evaluate the antitumor effect of these responses. We have combined the use of a predictive MHC class II binding peptide algorithm with in vitro vaccination of CD4 T cells using candidate peptides to identify naturally processed epitopes derived from LMP1 that elicit immune responses against EBV-expressing tumor cells. Peptide LMP1159-175 was effective in inducing HTL responses that were restricted by HLA-DR9, HLA-DR53, or HLA-DR15, indicating that this peptide behaves as a promiscuous T-cell epitope. Moreover, LMP1159-175–reactive HTL clones directly recognized EBV lymphoblastoid B cells, EBV-infected natural killer (NK)/T-lymphoma cells and naturally processed antigen in the form of LMP1+ tumor cell lysates presented by autologous dendritic cells. Because the newly identified epitope LMP1159-175 overlaps with an HLA-A2–restricted CTL epitope (LMP1159-167), this peptide might have the ability to induce simultaneous CTL and HTL responses against LMP1. Overall, our data should be relevant for the design and optimization of T-cell epitope–based immunotherapy against various EBV-associated malignancies, including NK/T cell lymphomas. [Cancer Res 2008;68(3):901–8]

Leukemias and lymphomas

Immune response to cancer

Tumor antigens

Cellular immunotheraphy

Cancer vaccines

Introduction

EBV is a B-lymphotrophic γ-herpes virus that is widespread in the human population and is associated with a number of malignancies, such as Hodgkin's lymphoma (HL), Burkitt's lymphoma, posttransplant lymphoproliferative disorder (PTLD), natural killer (NK)/T-cell lymphoma, and several lymphoepithelioma-like carcinomas, including nasopharyngeal carcinoma (NPC) and gastric carcinoma (
1–
4). In general, the majority of healthy individuals will not suffer from life-threatening disorders induced by EBV, because throughout life EBV-specific T lymphocytes control and inhibit the growth of EBV-infected or EBV-transformed cells (
5). Because MHC class I–restricted CTLs are considered the main effectors for providing protection against virus-associated malignancies, most researchers have focused their studies on characterizing CTL responses to EBV-derived latent and lytic cycle antigens (
6–
8).

In vitro established EBV-transformed B lymphoblastoid cell lines (EBV-LCL) and the EBV-induced proliferating cells found in PTLD are examples of the latency III infection stage. These cells express six nuclear antigens (EBNA1–EBNA6) and two latent membrane proteins (LMP1 and LMP2). The type II latency stage, in which only EBNA1 and LMP1 are expressed, is seen in more serious disorders, such as HL and NPC. Another more rare type of EBV infection/transformation has been observed in nasal NK/T cell lymphomas, which express EBNA1 and LMP1 in a latency II stage pattern.

LMP1 is required for the EBV-mediated transformation of B lymphocytes and has attracted significant interest because, thus far, it is the only EBV latency gene capable of transforming rodent fibroblasts in vitro, which are tumorgenic in nude mice (
9). Furthermore, it has been reported that B-cell lymphomas can spontaneously arise in LMP1 transgenic mice without the need of additional EBV genes (
10). In addition, LMP1 interacts with several signaling proteins of the tumor necrosis factor receptor family (
11) and Janus kinase 3 (
12), resulting in nuclear factor-κB (
13) and activator protein 1 (
14) induction and activation. Moreover, LMP1 protects against apoptosis by increasing bcl-2 activity (
15). In view of the above, LMP1 is considered as the main EBV-derived oncoprotein and has become an ideal target for T cell–based immunotherapy against EBV-induced malignancies.

For some time, immunotherapy for EBV-associated malignancies has been recognized as an attractive way to treat these diseases and eradicate EBV-transformed cells (
16). Most successful results have been obtained using adoptive T-cell immunotherapy with EBV-specific CTLs against HL and PTLD (
17–
19). In most instances, the EBV-reactive CTLs raised for adoptive immunotherapy were directed against the immunodominant EBNA3 antigen, which unfortunately is not expressed in HL, NPC, and NK/T cell lymphomas. Thus, to extend adoptive immunotherapy (or active immunization) against HL, NPC, and NK/T cell lymphomas it would be necessary to generate T-cell responses against other antigens, such as LMP1.

Although CTLs seem to play the major role in eradicating virus-infected cells, CD4 T-cell responses to viral infections are critical for the maintenance of virus-specific memory CTLs (
20). Moreover, CD4 T cells can exhibit a direct effector function against virus-infected or malignant cells that express MHC class II molecules (
21,
22). Indeed, it should be noted that many of the polyclonal T-cell preparations that were successful in treating EBV-PTLD in immunosuppressed individuals contained both CD4 and CD8 T cells (
16,
17,
23). Up to this date, there is limited information regarding the existence and frequency of MHC class II–restricted helper T-lymphocytes (HTL) responses against EBV latent antigens and the nature of the peptide epitopes capable of eliciting these responses (
24,
25). In addition, thus far, there has been no direct evidence that HTL can recognize LMP1-derived MHC class II epitopes on EBV-related tumor cells. Here, we report the identification of a novel peptide epitope derived from the EBV-LMP1 antigen that is capable of stimulating HTL responses in healthy individuals in the context of several MHC class II alleles. Most importantly, some of the peptide-induced HTLs directly recognized and killed EBV-infected NK cells isolated from patients suffering from chronic active EBV infection (CAEBV) and nasal NK/T cell lymphomas. We believe that our results will be of value for the development of immune therapies for EBV-associated malignancies, such as NK/T cell lymphoma, HL, and NPC.

Synthetic peptides. Potential HLA-DR–restricted CD4 T-cell epitopes were selected from the amino acid sequence of the EBV-LMP1 protein using the peptide/MHC binding prediction algorithm tables derived for three HLA-DR alleles (DRB1*0101, DRB1*0401, and DRB1*0701) as described by Southwood et al. (
29). The predicted peptide epitopes were synthesized by solid-phase organic chemistry and purified by high-performance liquid chromatography (HPLC). The purity (>80%) and identity of peptides were assessed by HPLC and mass spectrometry, respectively.

In vitro induction of antigen-specific HTLs with synthetic peptides. The procedure used for the generation of LMP1-reactive HTL lines using peptide-stimulated lymphocytes has been described in detail (
30). Briefly, dendritic cells were produced in tissue culture from monocytes (purified with anti-CD14 antibody-coated magnetic microbeads; Miltenyi Biotech) that were cultured for 7 days at 37°C in a humidified CO2 (5%) incubator in the presence of 50 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) and 1,000 IU/mL interleukin-4 (IL-4). Peptide-pulsed dendritic cells (3 μg/mL for 2 h at room temperature) were irradiated (4,200 rad) and cocultured with autologous purified CD4+ T cells in 96–round-bottomed well culture plates. One week later, the CD4 T cells were restimulated in individual microcultures with peptide-pulsed irradiated autologous PBMC, and 2 days later, human rIL-2 was added at a final concentration of 10 IU/mL. One week later, the T cells were tested for antigen reactivity using a cytokine-release assay as described below. Those microcultures exhibiting a significant response of cytokine-release to peptide (at least 2.5-fold over background) were expanded in 24-well or 48-well plates by weekly restimulation with irradiated peptide-pulsed autologous PBMC. Complete culture medium for all procedures consisted of AIM-V (Invitrogen/Life Technologies) supplemented with 3% human male AB serum. All blood samples were obtained after the appropriate informed consent.

Measurement of antigen-specific responses with HTL lines. CD4 T cells (3 × 104 per well) were mixed with irradiated APCs in the presence of various concentrations of antigen (peptides or tumor lysates) in 96-well culture plates. APCs consisted of either autologous PBMC (1 × 105 per well), HLA-DR–expressing L cells (3 × 104 per well), MHC-typed EBV-LCL (3 × 104 per well), autologous dendritic cells (5 × 103 per well), and EBV-transformed NK cell lines KAI3 or SNT8. Tumor cell lysates were prepared by three freeze-thaw cycles of 1 × 108 cells resuspended in 1 mL of serum-free RPMI 1640. Cell lysates were used as a source of antigen at a concentration of 5 × 105 cell equivalents per milliliter. Culture supernatants were collected after 48 h for measuring antigen-induced lymphokines (IFN-γ, GM-CSF, IL-10) production using commercial ELISA kits (BD PharMingen). To show antigen specificity and MHC restriction, blocking of antigen-induced responses was assessed by adding anti–HLA-DR monoclonal antibody (mAb) L243 (IgG2a; prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or anti–HLA-A/B/C mAb W6/32 (IgG2a; ATCC) at 10 μg/mL throughout the 48-h incubation period. All ELISA determinations were carried out in triplicate samples, and results correspond to the mean values with SD of the mean.

Cell-mediated cytotoxicity assays. Cytotoxic activity of HTLs was measured using in a colorimetric CytoTox 96 assay (Promega). This system quantifies the release of lactate dehydrogenase (LDH) from target cells. T cells were mixed with 2 × 104 targets at different effectors to target (E:T) ratios in 96–round-bottomed well plates. After 6 to 9 h of incubation at 37°C, a 50-μL sample of supernatant was collected from each well to measure LDH content. To correct for spontaneous LDH release from effector cells, LDH levels were measured for each individual effector cell concentration used in the experimental set up (effector spontaneous). All measured values were assayed in triplicate and corrected for the culture medium LDH background. The percentage of specific LDH release was determined as percentage cytotoxicity = [(experimental − effector spontaneous − target spontaneous) / (target maximum − target spontaneous)] × 100.

Results

Selection of potential helper T-cell epitopes from EBV-LMP1. As with previous studies (
22,
30–
36), an MHC class II peptide binding predictive algorithm (
29) was used to select candidate peptides from the LMP1 sequence that would bind to the products of three common human MHC class II alleles (HLA-DR1, HLA-DR4, and HLA-DR7). Three peptides, LMP135-49 (ALLFWLYIVMSDWTG), LMP1102-116 (GQALFLGIVLFIFGC), and LMP1159-175 (YLQQNWWTLLVDLLWLL) were identified as potentially binding the three MHC class II alleles (data not presented), suggesting these could function as promiscuous HTL epitopes. In past studies, we have observed that peptide sequences predicted to bind to HLA-DR1, HLA-DR4, and HLA-DR7 have elicited HTL responses restricted by additional MHC class II alleles, such as DR9, DR13, DR15, or DR53 (
22,
30,
32,
34–
36).

Generation of HTLs specific for LMP1 peptides. The three selected LMP1 peptides were synthesized and tested for their ability to stimulate purified CD4 T cells obtained from five healthy EBV-seropositive donors in primary in vitro cultures using peptide-pulsed autologous dendritic cell as APC. The MHC class II alleles expressed by these individuals were donor 1 (DR4/9, DR53), donor 2 (DR4/15, DR51/53), donor 3 (DR1/15, DR51), donor 4 (DR9/14, DR52/53), and donor 5 (DR9/14, DR52/53). After three cycles of stimulation (using γ-irradiated peptide-pulsed autologous PBMC as APC for the second and third stimulations), only one of the three peptides (LMP1159-175) was able to elicit antigen-reactive HTL responses in two of the five donors (donor 3 and donor 4; data not shown). Several T-cell clones were isolated to study the fine specificity and MHC restriction patterns of these responses. As shown in
Fig. 1
, all four HTL clones, K23 and SK7 (from donor 3) and clones MT6B and MT7 (from donor 4), responded to peptide LMP1159-175 presented by autologous PBMC in a dose-dependent manner. To show that HLA class II molecules presented peptide LMP1159-175 to the HTL clones, we measured the T-cell recognition of antigen in the presence of antibodies against HLA class I molecules (W6/32) or HLA-DR molecules (L243). In all cases, the antigen response to autologous PBMC presenting peptide was blocked by anti–HLA-DR mAb L243 but not by anti-HLA class I mAb W6/32 (
Fig. 2
). These results indicate that the recognition of peptide LMP1159-175 by the HTL clones was restricted by HLA-DR molecules, because mAb L243 reacts only with HLA-DR and not with HLA-DQ or HLA-DP. In addition, when transfected mouse fibroblasts (L cells) expressing individual HLA-DR molecules were used as APCs, we observed that peptide-pulsed DR15-transfected L cells (L-DR15) stimulated HTL clones K23 and SK7, L-DR9 cells presented peptide to HTL clone MT6B, and L-DR53 cells presented peptide to HTL clone MT7 (
Fig. 2). These results indicate that LMP1159-175 behaves as a classic promiscuous HTL epitope because it can be presented to T cells by more than one MHC class II allele. To further evaluate the frequency of HTL responses to LMP1159-175 in normal individuals (presumably seropositive for EBV), we stimulated in short-term cultures PBMC from an additional 10 blood donors with this peptide epitope. The results indicated that 8 of the 10 donors exhibited significant T-cell responses to peptide LMP1159-175 (Supplementary Fig. S1). Interestingly, two of the donors that responses to peptide LMP1159-175 in this experiment did not express DR9, DR15, nor DR53, suggesting that the MHC class II promiscuity of HTL epitope LMP1159-175 may go beyond these three alleles.

Recognition of peptide LMP1159-175 by CD4+ T cells. HTL clones K23, SK7, MT6B, and MT7 were tested for their capacity to recognize autologous PBMC as APCs in the presence of various concentrations of peptide LMP1159-175. Points, mean of triplicate determinations; bars, SD. Points without bars had SD of <10% the value of the mean. Results are representative of at least two experiments that were done with the same samples.

MHC restriction analysis of LMP1159-175-reactive HTL. Assessment of the capacity of inhibition of anti-DR mAb L234 (αDR) or anti–MHC class I mAb W6/32 (αClass I) to inhibit peptide (LMP1159-175)–induced production of IFN-γ when using PBMC as APC. In addition, responses of the peptide LMP1159-175 were evaluated using L cells transfected with individual HLA-DR genes as APCs to define the restriction MHC class II molecules. Columns, mean of triplicate determinations; bars, SD. Columns without bars had SD of <10% the value of the mean. Results are representative of at least two experiments that were done with the same samples.

Peptide LMP1159-175–reactive HTLs recognize EBV-transformed cells. Next, we evaluated whether LMP1159-175–reactive HTLs had the capacity to recognize the naturally processed viral antigen expressed by EBV-infected or EBV-transformed lymphocytes. However, before examining the ability of direct recognition of LMP1-expressing transformed cells by peptide-elicited HTLs, we first characterized the MHC class II alleles and the expression levels of LMP1 protein in several EBV-LCLs and EBV-expressing NK/T cell lymphoma cell lines to be used as APCs in the HTL stimulation assays. The expression of LMP1 protein was observed in EBV-positive NK cell lymphoma line (KAI3), EBV-postive NK/T cell lymphoma lines (SNK6 and SNT8), EBV-LCLs (LCL-MT, Ky, and Wa), and in the Burkitt's lymphoma Raji, but not in the EBV-negative Jurkat T lymphoma cell line (Supplementary Fig. S2A). The cell surface expression of MHC class II molecules was evaluated by flow cytometry to select those LMP1-expressing cell lines that could directly stimulate the LMP1-specific HTL clones. EBV-positive NK cell lymphomas KAI3 (DR9/DR53) SNT8 (DR15), EBV-LCLs, and Raji cell (DR3/DR10) expressed high levels of MHC class II molecules, whereas the EBV-negative Jurkat T lymphoma cell line (to be used as a negative control) did not (Supplementary Fig. S2B).

With this information on hand, we proceeded to test the capacity of LMP1159-175–reactive HTLs to recognize LMP1-expressing cells with matching MHC class II alleles. The results presented in
Fig. 3
show that HTL clone SK7 (DR15 restricted), clone MT6B (DR9 restricted), and clone MT7 (DR53 restricted) were all effective in recognizing autologous EBV-LCLs and MHC class II–matched EBV-LCLs, but were unable to respond to MHC class II–mismatched EBV-LCLs or the MHC class II–negative Jurkat T-cell lymphoma. Furthermore, recognition of EBV-LCLs by the HTLs was inhibited by the addition of anti–HLA-DR mAb L243, confirming that the peptide epitope was recognized through MHC class II molecules. On the other hand, HTL clone K23 (DR15 restricted) was unable to recognize autologous or MHC class II–matched EBV-LCLs (data not shown).

Assessment of naturally processed LMP1 antigen expressed in EBV-LCL by LMP1159-175–reactive CD4+ HTL clones. Various EBV-LCL lines with matching or with unrelated HLA-DR molecules were tested for the capacity to directly stimulate antigen responses of HTL clones SK7, MT6B, and MT7. Jurkat cells (EBV-negative) were used as negative controls. Antigen-specificity of these responses was evaluated by blocking the production of IFN-γ using anti–HLA-DR mAb L243. Columns, mean of triplicate determinations; bars, SD. Columns without bars had SD of <10% the value of the mean. Results are representative of two experiments that were done with the same samples.

The above results indicate that EBV-LCLs, functioning as latency III stage–infected/transformed cells, are able to present the naturally processed LMP1159-175 epitope on their MHC class II molecules to antigen-specific HTL clones. Because we were more interested in the use of LMP1 as a target for immunotherapy for latency II malignancies, such as NK/T cell lymphomas, HTL clone SK7 was tested for its ability to directly recognize the NK/T cell lymphoma SNT8 (DR15). At the same time, HTL clones MT6B and MT7 were tested for their ability to directly recognize the EBV-infected NK cell lymphoma KAI3, which expresses the MHC class II restriction elements used by these clones (DR9 and DR53). As shown in
Fig. 4A
, clone SK7 reacted with the NK/T cell lymphoma SNT8 and clones MT6B and MT7 were effective in directly recognizing the KAI3 lymphoma cells. In all three cases, recognition of the NK, NK/T cell lymphomas by the HTL was inhibited by anti–HLA-DR mAb L243 (
Fig. 4B), but not by anti–MHC class I mAb (data not shown). Overall, these results show that the LMP1-specific HTL can directly recognize antigen on EBV-LCL and NK, NK/T cell lymphomas and validate that LMP1, and specifically the LMP1159-175 HTL epitope, can serve as a therapeutic target in malignant cells of the latency type III or type II of EBV infection.

Recognition of EBV carrying NK cell lymphoma by peptide LMP1159-175–specific HTL clones. A, HTL clone SK7 (top) was tested for its ability to respond to various numbers of SNT8 NK/T lymphoma cells or Raji (negative control). MT6B and MT7 HTL clones (middle and bottom, respectively) were tested for their ability to respond to the KAI3 infected NK cell line or Raji (negative control). Points, mean of triplicate determinations; bars, SD. Points without bars had SD of <10% the value of the mean. B, specificity of recognition of LMP1-expressing tumor cells by HTLs was evaluated by the capacity of anti–HLA-DR mAb L243 to block the production of IFN-γ. Autologous ENV-LCL and Raji cells were included as controls. Columns, mean of triplicate determinations; bars, SD. Columns without bars had SD of <10% the value of the mean. Results are representative of two similar experiments.

Cytotoxic activity of peptide LMP1159-175–specific HTL. It has been observed that MHC class II–restricted CD4+ T lymphocytes can behave as effector cells, sometimes being capable of killing viral-infected and tumor cells. Thus, we evaluated whether the peptide LMP1159-175–specific HTL clones had the capacity to kill the EBV-infected lymphocytes, such as EBV-LCL and EBV-positive NK cells. As shown in
Fig. 5
, the DR15-restricted clone SK7 was effective in killing autologous EBV-LCL and the NK/T cell lymphoma SNT8, but not Raji cells. Similarly, the DR53-restricted HTL clone MT7 exhibited high cytolytic activity against the autologous EBV-LCL and the EBV-positive NK cell lymphoma KAI3 in the absence of exogenously added peptide, but not toward the DR53-negative, LMP1-positive Raji Burkitt's lymphoma cells. On the other hand, the DR9-restricted HTL clone MT6B did not display any significant cytotoxic activity against EBV-positive tumor cells. As with previous experiments, antibodies to MHC class II (anti–HLA-DR mAb L243), but not to MHC class I, effectively inhibited (up to 80%) the cytolytic response to the EBC-LCLs, NK/T cell lymphoma, and EBV-positive NK cells (data not shown), indicating that antigen recognition is mediated through the recognition of peptide in the context of MHC class II molecules. Furthermore, the capacity of the anti–MHC class II antibodies to block the cell-mediated killing indicates that this cytotoxicity is mostly unidirectional and that the NK/T cells or the EBV-positive NK cells are not killing the CD4 T-cell clones.

Assessment of cytolytic activity of LMP1159-175–specific HTL clones. The capacity of LMP1-reactive CD4+ T cells to kill EBV-infected NK/T, NK, or LCLs was evaluated as described in Materials and Methods. Points, mean of triplicate determinations. Points without bars had SD of <10% the value of the mean.

Recognition of naturally processed EBV-LMP1 antigen presented by professional APC. Lastly, we evaluated whether professional APC, such as dendritic cell, would be able to capture and process antigens derived from dead LMP1-expressing tumor cells (cell lysates) and present the LMP1159-175 epitope to antigen-specific HTLs. The results in
Fig. 6
show that all three HTL clones, MT6B, MT7, and SK7, were capable of recognizing antigen prepared from lysates from EBV-LCL, EBV-negative NK/T cell lymphomas, or EBV-positive NK cells and that these responses were inhibited by the addition of anti–HLA-DR mAbs. On the other hand, as expected, little reactivity was evident when the HTL were stimulated with dendritic cell alone or dendritic cell pulsed with EBV-negative Jurkat cell lysate. These results indicate that professional APCs are effective in processing LMP1 antigen and presenting the LMP1159-175 epitope to HTL in the context of HLA-DR9, DR15, and DR53. HTL clone K23 (DR15 restricted), which as previously mentioned did not recognize antigen presented directly by MHC class II–positive EBV-infected/transformed cells, was also not able to recognize tumor cell lysates presented by dendritic cell (data not shown).

Presentation of naturally processed LMP1159-175 epitope to HTL by dendritic cells (DC). Autologous dendritic cells were pulsed with cell lysates or LMP1159-175 peptide and then tested for their ability to stimulate HTL clones SK7, MT6B, and MT7. Jurkat T-cell lymphoma lysate was used as negative control and peptide-pulsed dendritic cell as positive control. The capacity of anti-DR mAb L243 was assessed to determine that T-cell reactivity was through TCR/MHC interactions. Columns, mean of triplicate determinations; bars, SD. Columns without bars had SD of <10% the value of the mean. Results are representative of at least two experiments.

Discussion

EBV-associated malignancies, such as HL, NPC, and NK/T cell lymphoma, belonging to the latency type II of infection, are clear examples of human diseases that require more efficient therapeutic strategies, such as immunotherapy. Because EBV-positive HL and peripheral NK/T cell lymphomas often occur in individuals with uncompromised basal immunity (
37), these diseases are an attractive target for T cell–based immunotherapy. EBNA1 and LMP1 antigens have been considered as prime candidates for developing T-cell immunotherapies for EBV latency II malignancies. A large number of MHC class I–restricted and MHC class II–restricted T-cell epitopes from EBNA1 have been identified (
38,
39). On the other hand, few CTL epitopes for LMP1 have been reported (
40–
42), and the availability of MHC class II–restricted HTL epitopes for this antigen is even more scarce. Leen et al. (
24) described three peptides (LMP1130-144, LMP1212-226, and LMP1340-354) and Depil et al. (
25) reported one peptide (LMP168-83) that were recognized by human CD4 T cells. However, detailed MHC restriction analyses and evidence of T-cell recognition of EBV-transformed cells was not presented. Here, we show for the first time that the LMP1 antigen is processed and presented to CD4 HTLs in the context of HLA class II molecules through the endogenous (direct presentation by transformed cells) or the exogenous (antigens captured and processed by conventional APC) pathways. In our studies, three peptides (LMP135-49, LMP1102-116, and LMP1159-175), which did not correspond to those described by Leen and Depil (
24,
25) were predicted to potentially function as promiscuous HTL epitopes. Out of the three peptides, only one, LMP1159-175, proved to be effective at inducing HTL responses. Our lack of success at eliciting peptide-reactive HTLs to the other two candidate peptides could be due to the presence of low precursor frequencies of T cells specific for LMP1 protein in peripheral blood (
6). Furthermore, there is some evidence that the LMP1 protein contains a motif that impairs T-cell responses, lowering the frequency of LMP1-reactive HTL in individuals with previous contact with EBV (
43). In agreement with this, it has been shown that anti-LMP1 antibodies in sera from healthy virus carriers are not detectable or are present at low titers, indicating that LMP1 is not very immunogenic, perhaps because it is not efficiently processed by APCs (
44,
45). On the other hand, there is a report of significant amount of antibodies to LMP1 in sera of patients with EBV-associated NPC (
46), suggesting that the immunogenic potential of this antigen may vary depending on the type of disease. Another potential explanation to our inability to generate HTL responses to peptides LMP135-49 and LMP1102-116 is that these peptides may not bind with sufficient strength to the MHC class II alleles expressed by the blood donors used in the present study. Although we cannot exclude the possibility that LMP135-49 and LMP1102-116 may function as HTL epitopes, our studies show that LMP1159-175 was capable of stimulating HTL responses in several individuals in the context of at least three MHC class II alleles and that the resulting HTLs were effective in recognizing naturally processed antigen.

The importance of CD4 T lymphocytes for the induction, expansion, and maintenance of antitumor CTL responses is generally accepted. For example, work done in our laboratory has shown that HTL can facilitate the expansion of CTL by direct costimulation through CD70/CD27, 4-1BBL/4-1BB interactions and by preventing CTL activation–induced cell death (
47,
48). However, other subsets of CD4 T lymphocytes, known as regulatory T cells (Treg) are known to inhibit CTL responses (
49). Marshall et al. (
50) reported that APC presenting LMP1 protein or synthetic peptides from LMP1 stimulated CD4 T cells from EBV-seropositive donors to produce significant amounts of IL-10 and to inhibit a variety of T-cell responses. It should be noted that the promiscuous HTL epitope described here (LMP1159-175) did not correspond to any of the peptides reported to generate IL-10–producing CD4+ T cells (
50). In addition, although we did not test whether the LMP1159-175–reactive HTL produced IL-10 in response to antigen stimulation, our results show that these CD4+ T cells secreted abundant IFN-γ, indicating that these cells are not typical Treg lymphocytes. Nevertheless, we cannot exclude the possibility that, under certain circumstances, LMP1159-175–reactive CD4+ T cells could exhibit Treg function.

An interesting observation is that the HTL epitope LMP1159-175 contains, within its sequence, a previously described (
41) HLA-A2–restricted CTL epitope LMP1159-167 (YLQQNWWTL). Thus, one could envision that administration of peptide LMP1159-175 as a vaccine would have the potential of simultaneously inducing both CTLs and HTL with effector antitumor activity in individuals expressing HLA-A2 and one of the MHC class II alleles capable of presenting this peptide. Such a vaccine could prove to be of value, for example, in the prevention of extranodal nasal NK/T cell lymphomas, which are aggressive lymphoproliferative disorders of EBV-infected NK/T cells occurring in individuals with CAEBV. Although the course of CAEBV is relatively long, most of patients ultimately develop NK cell leukemia/lymphoma, which tend to be resistant to chemotherapy, presenting a poor prognosis to these patients. A therapeutic peptide-based vaccine capable of stimulating both CD4 and CD8 effector T cells to an EBV antigen expressed during latency II infection, such as LMP1, may be able to inhibit or delay the progression to NK cell leukemia/lymphoma in patients with CAEBV. This therapeutic approach could also be applicable to other latency type II malignancies, such as HL and NPC. In those circumstances, wherein patients may not respond to vaccination due to immune compromised state induced by their malignancy, one could envision the in vitro generation and expansion of CD4 and CD8 T-cell lines using peptide LMP1159-175 for the use of adoptive immunotherapy.

Acknowledgments

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.